Current recombinant expression strategies in host cells such as E. coli, insect cell culture, and mammalian cell culture express and secrete proteins at very high level in the culture media. Using these systems high levels of expression, proper protein folding and post-translational modification of proteins, is achieved. Furthermore, purification of the expressed protein is simplified since intracellular proteins may be readily segregated from other components (DNA, vesicle, membranes, pigments, and so on). For plant or yeast expression systems, the cell wall prevents secretion of expressed protein into the culture media.
Different approaches are widely used in science to generate cell-extracts. Mechanical approaches to disrupt cell wall and liberate its content are not usually selective for certain class of protein or cellular components. Directing expression of a protein of interest into the cell culture media, allowing intracellular contaminants to be removed by centrifugation or by filtration, allow simple and fast enrichment of the protein of interest. It may also be desirable to separate a protein or a protein suprastructure of interest, including protein rosettes, nanoparticles, large protein complexes, antibodies or virus-like particles (VLPs), and the like, from some, or all of the proteins, DNA, membrane fragments, vesicles, pigments, carbohydrates, etc. present in the plant or plant matter before the protein or protein suprastructure of interest is used in vaccine formulation.
Immunoglobulins (IgGs) are complex heteromultimeric proteins with characteristic affinity for specific antigenic counterparts of various natures. Today, routine isolation of IgG-producing cell lines, and the advent of technologies for IgG directed evolution and molecular engineering have profoundly impacted their evolution as biotherapeutics and in the general life science market. Therapeutic monoclonal IgG (monoclonal antibodies, mAbs) dominate the current market of new anti-inflammatory and anti-cancer drugs and hundreds of new candidates are currently under research and clinical development for improved or novel applications. The annual market demand for mAbs ranges from a few grams (diagnostics), a few kilograms (anti-toxin) to up to one or several hundreds of kilograms (bio-defense, anti-cancer, anti-infectious, anti-inflammatory). Methods to produce modified glycoproteins from plants is described in WO 2008/151440 (which is incorporated herein by reference).
A method for extracting protein from the intercellular space of plants, comprising a vacuum and centrifugation process to provide an interstitial fluid extract comprising the protein of interest is described in PCT Publication WO 00/09725 (to Turpen et al.). This approach is suitable for small proteins (of 50 kDa or smaller) that pass through network of microfibers under vacuum and centrifugation, but is not suitable for larger proteins, superstructure proteins, protein rosettes, nanoparticles, large protein complexes, such as antibodies or VLPs.
McCormick et al 1999 (Proc Natl Acad Sci USA 96:703-708) discloses use of a rice amylase signal peptide fused to a single-chain Fv (scFv) epitope to target the expressed protein to the extracellular compartment, followed by vacuum infiltration of leaf and stem tissue for recovery of the scFv polypeptides. Moehnke et al., 2008 (Biotechnol Lett 30:1259-1264) describes use of the vacuum infiltration method of McCormick to obtain a recombinant plant allergen from tobacco using an apoplastic extraction. PCT Publication WO 2003/025124 (to Zhang et al) discloses expression of scFv immunoglobulins in plants, targeting to the apoplastic space using murine signal sequences.
Virus-like particles (VLPs) may be employed to prepare vaccines, such as influenza vaccines for example. Suprastructures such as VLPs mimic the structure of the viral capsid, but lack a genome, and thus cannot replicate or provide a means for a secondary infection. VLPs offer an improved alternative to isolated (soluble) recombinant antigens for stimulating a strong immune response. VLPs are assembled upon expression of specific viral proteins and present an external surface resembling that of their cognate virus but, unlike true viral particle, do not incorporate genetic material. The presentation of antigens in a particulate and multivalent structure similar to that of the native virus achieves an enhanced stimulation of the immune response with balanced humoral and cellular components. Such improvement over the stimulation by isolated antigens is believed to be particularly true for enveloped viruses as enveloped VLPs present the surface antigens in their natural membrane-bound state (Grgacic and Anderson, 2006, Methods 40, 60-65). Furthermore, influenza VLPs, with their nanoparticle organization, have been shown to be better vaccine candidates compared to recombinant hemagglutinin HA (i.e. monomeric HA, or HA organized into rosettes; assembly of 3-8 trimers of HA), and they are able to activate both humoral and cellular immune response. (Bright, R. A., et. al., 2007, Vaccine 25, 3871-3878).
The production of influenza HA VLPs that comprise a lipid envelope has been previously described by the inventors in WO 2009/009876 and WO 2009/076778 (to D'Aoust et al.; both of which are incorporated herein by reference). For enveloped viruses, it may be advantageous for a lipid layer or membrane to be retained by the virus. The composition of the lipid may vary with the system (e.g. a plant-produced enveloped virus would include plant lipids or phytosterols in the envelope), and may contribute to an improved immune response.
The assembly of enveloped VLPs in transgenic tobacco expressing the HBV surface antigen (HBsAg) was described by Mason et al. (1992, Proc. Natl. Acad. Sci. USA 89, 11745-11749). Plant-produced HBV VLPs were shown to induce potent B- and T-cell immune responses in mice when administered parenterally (Huang et al., 2005, Vaccine 23, 1851-1858) but oral immunization through feeding studies only induced a modest immune response (Smith et al., 2003, Vaccine 21, 4011-4021). Greco (2007, Vaccine 25, 8228-8240) showed that human immunodeficiency virus (HIV) epitopes in fusion with HBsAg accumulated as VLP when expressed in transgenic tobacco and Arabidopsis, creating a bivalent VLP vaccine.
Expression of the viral capsid protein (NVCP) in transgenic tobacco and potato plants resulted in the assembly of non-enveloped VLPs (Mason et al., 1996, Proc. Natl. Acad. Sci. USA 93, 5335-5340). NVCP VLPs have been produced in agroinfiltrated N. benthamiana leaves (Huang et al. 2009, Biotechnol. Bioeng. 103, 706-714) and their immunogenicity upon oral administration demonstrated in mice (Santi et al., 2008, Vaccine 26, 1846-1854). Administration of 2 or 3 doses of raw potatoes containing 215-751 μg of NVCP in the form of VLPs to healthy adult volunteers resulted in development of an immune response in and 95% of the immunized volunteers (racket et al. 2000, J. Infect. Dis. 182, 302-305). Non-enveloped VLPs have also been obtained from the expression of HBV core antigen (HBcAg; Huang et al., 2009, Biotechnol. Bioeng. 103, 706-714), and the human papillomavirus (HPV) major capsid protein L1 (Varsani et al., 2003, Arch. Virol. 148, 1771-1786).
A simpler protein, or protein suprastructure production system, for example, one that relies on the expression of only one or a few proteins is desirable. Production of proteins, or protein suprastructures, for example but not limited to protein rosettes, nanoparticles, large protein complexes such as antibodies or VLPs, in plant systems is advantageous, in that plants may be grown in a greenhouse or field, and do not require aseptic tissue culture methods and handling.
Methods of recovering proteins or protein suprastructures that are substantially free of, or easily separated from plant proteins, yet retain the structural and characteristics of the proteins or protein suprastructures are desired.